I've been studying field hippocampal electrophysiology in mice for many years and we've recently started working in rats. I am beyond frustrated because things do not work at smoothly in rats and I don't seem to get great LTP with either tbs of 100Hz HFS. I've been scouring the literature for in vitro rat Schaffer collateral electrophysiology and techniques are all over the place.
I'll tell you about my setup and perhaps you can suggest alterations?
400 um horizontal sections from vibratome; stored for 20 minutes in 50/50 Sucrose cutting solution/ACSF for 20 minutes before transferring to chamber
Interface brain slice chamber from Automate Scientific. Flow rate 1 ml/min (this is what we do for mice but I see most rat is a higher flow rate)
Slices maintained at 30 degrees C with oxygenated 95%/5% ACSF for 1.5-2 hours before recording.
I use bipolar nichrome wire stimulating electrodes, 1-4mOhm resistance recording electrodes.
I stimulate at the end of CA3 and record in the mid region of CA1.
I see some people remove CA3 completely. Should I do that?
Any help would be greatly appreciated.
Hi Melinda,
I worked with rat slices for many years, and have more recently started working with mouse slices, so my experience is the inverse of yours!
Although I would do things a little differently (see below), I think your procedures are probably fine overall. However, the main thing that jumps out to me is that you are making horizontal slices, which will give you slices from the ventral hippocampus. As you may be aware, there are several publications showing a large difference in LTP between dorsal and ventral hippocampus, with LTP being much smaller, or even absent, in the ventral (a couple of papers linked here). My anecdotal observations since using mouse slices is that this dorsal/ventral difference in plasticity is much less pronounced in the mouse than the rat, so it could simply be a species difference that you are seeing. Might be worth trying some dorsal (sagittal) rat slices to see what sort of LTP you get all else being equal.
Other than that, here are some small differences between your procedures and mine which may or may not make a difference:
- I cut in sucrose (full sucrose, no NaCl) and then transfer straight to normal ACSF at room temp for recovery (recordings done at ~30C). I warm the slices to 30C for 20 min after slicing only when making slices for patch clamp from adult animals.
- I usually do submerged recordings, but no reason why interface should be a problem. Flow rate of at least 2 ml/min has always been pretty standard in my experience (maybe your slices aren't quite getting enough oxygen at 1 ml/min?... though this should be less of a problem with interface in theory).
- I usually cut off the CA3, though I've known others who prefer to leave it on. Shouldn't make too much difference, unless perhaps you are using conditions which make the slices more excitable, in which case having the CA3 on might increase the chances of unwanted spontaneous network activity (which could in turn affect LTP induction).
- Rat slices are much bigger than mouse slices (obviously). So it could be that your nichrome wire stim electrodes (which I'm assuming are quite 'thin') are not stimulating, proportionally, the same number of fibres in the rats (i.e. the relative area of stratum radiatum stimulated in rats would be less than in mice). This could potentially mean less postsynaptic firing in response to stimulation, which could reduce the likelihood of inducing LTP. I used to use the concentric bipolar electrodes from FHC for rat slices, stimulating at the CA3 end of the slice on the CA1 side of the CA1/CA2 border.
Hope that helps a bit! Good luck.
Article Decreased ability of rat temporal hippocampal area CA1 to pr...
Article Unique regulation of long term potentiation in the rat ventr...
How do you choose your stimulation intensity? Also, can you show us some waveforms?
There is one older study which suggested that slices prepared in high sucrose aCSF displayed reduced LTP. So you could try altering the cutting medium and store directly in aCSF rather than in 50:50 sucrose.
Article Reduced long-term potentiation in hippocampal slices prepare...
Hi Melinda,
I worked with rat slices for many years, and have more recently started working with mouse slices, so my experience is the inverse of yours!
Although I would do things a little differently (see below), I think your procedures are probably fine overall. However, the main thing that jumps out to me is that you are making horizontal slices, which will give you slices from the ventral hippocampus. As you may be aware, there are several publications showing a large difference in LTP between dorsal and ventral hippocampus, with LTP being much smaller, or even absent, in the ventral (a couple of papers linked here). My anecdotal observations since using mouse slices is that this dorsal/ventral difference in plasticity is much less pronounced in the mouse than the rat, so it could simply be a species difference that you are seeing. Might be worth trying some dorsal (sagittal) rat slices to see what sort of LTP you get all else being equal.
Other than that, here are some small differences between your procedures and mine which may or may not make a difference:
- I cut in sucrose (full sucrose, no NaCl) and then transfer straight to normal ACSF at room temp for recovery (recordings done at ~30C). I warm the slices to 30C for 20 min after slicing only when making slices for patch clamp from adult animals.
- I usually do submerged recordings, but no reason why interface should be a problem. Flow rate of at least 2 ml/min has always been pretty standard in my experience (maybe your slices aren't quite getting enough oxygen at 1 ml/min?... though this should be less of a problem with interface in theory).
- I usually cut off the CA3, though I've known others who prefer to leave it on. Shouldn't make too much difference, unless perhaps you are using conditions which make the slices more excitable, in which case having the CA3 on might increase the chances of unwanted spontaneous network activity (which could in turn affect LTP induction).
- Rat slices are much bigger than mouse slices (obviously). So it could be that your nichrome wire stim electrodes (which I'm assuming are quite 'thin') are not stimulating, proportionally, the same number of fibres in the rats (i.e. the relative area of stratum radiatum stimulated in rats would be less than in mice). This could potentially mean less postsynaptic firing in response to stimulation, which could reduce the likelihood of inducing LTP. I used to use the concentric bipolar electrodes from FHC for rat slices, stimulating at the CA3 end of the slice on the CA1 side of the CA1/CA2 border.
Hope that helps a bit! Good luck.
Article Decreased ability of rat temporal hippocampal area CA1 to pr...
Article Unique regulation of long term potentiation in the rat ventr...
Hi Melinda,
I think you could try NMDG instead of sucrose, if you want to see nice LTP. Look at the papers of Jonathan Ting for comparison.
I don't think you need to excise the CA3, it only makes a difference if you are using GABAA blockers above a certain concentration (for bicuculline seems to be 5 micromolar) because the CA3 neurons start firing spontaneously and it would be difficult to properly record. Other than that, no need to cut the Schaffer collaterals.
Good luck!
Thank you, Patrick Tidball . I was mistaken and actually do saggital sections. I forgot my anatomy briefly! I think I will try the 4 wire electrodes that I have stored away. I normally use only 2 (which is perfect for mouse). Like I say, I get stable signals but no potentiation which may be due to not enough neurons being fired.
Thanks again!
Alexander F Hoffman , that is very interesting! I recently discovered that my glucose (yes, I know, not sucrose) is very high in my ACSF. I actually think I will try to prepare my next rat brain in ice cold ACSF instead in light of this strange news.
William M Connelly I do not have my Clampfit file for rat with me currently to show you a waveform. Will post another time. I stimulate at 1/2 maximum fEPSP attained through an input/output curve. Does that help?
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